JP2011521637A - Monitoring and therapeutic delivery of targeted reporter fusion proteins using gene and optical imaging - Google Patents

Abstract

  The present invention relates to a method for noninvasively monitoring the expression of a gene of interest in a cell when the cell is contacted with a compound that affects the expression of the gene of interest. The present invention also relates to a variety of isolated nucleic acid molecules having coding sequences. The coding sequence has a gene of interest sequence encoding a gene of interest polypeptide fused to a reporter sequence encoding a fluorescent reporter polypeptide and is operably linked to a promoter sequence. The present invention also delivers a compound that affects the expression of the gene of interest in the cell and not only monitors the effect of the expression of the gene of interest in the subject induced by the compound, but also monitors the delivery of the compound simultaneously. It also relates to the use of the nucleic acid molecules and methods of the invention to do so.

Description

  The present invention relates to a method for noninvasively monitoring the expression of a gene of interest in a cell when the cell is contacted with a compound that affects the expression of the gene of interest.

  Thus, the present invention relates to a method for monitoring an increase in the expression of a gene of interest in a cell when contacting the cell with a demethylating agent. The present invention also relates to monitoring a decrease in expression of a gene of interest in a cell when contacting the cell with RNAi. The method is performed inside or outside the human or animal body.

  The invention also relates to different nucleic acid molecules having sequences encoding the fluorescent reporter polypeptide and the gene of interest polypeptide.

  One of the major tasks of today's drugs is the development of therapeutic systems that can selectively affect the expression of genes that are suspected of being mis-adjusted or known to be mis-adjusted. is there. Depending on the gene affected, this manifests a serious disease, for example cancer. Many efforts have been made in this area, but there is a need to improve the results obtained so far and to combine the results with a system that can easily monitor, for example, the effectiveness of treatment and / or the delivery of therapeutic compounds. Still there.

  In general, the concept of gene expression can be summarized including several steps, at the DNA level, which are coding with specific sequences for proteins or RNAs that both exhibit specific functions. In the first step, DNA is transcribed into the corresponding RNA. This step is tightly controlled by including so-called promoter regions, often near the coding sequence at the 5 'end, as well as transcription factors that positively and negatively affect transcription and further upstream or downstream regulatory DNA elements. Is done. If the protein is the final product of gene expression, the transcribed RNA is called mRNA. In the second step, this mRNA is translated into a protein, which is further optionally post-translationally modified in subsequent steps. If RNA is the final product, the transcribed RNA is not only included in a complex with, for example, a protein, but is rearranged and processed. Thus, for example, it is possible to influence gene expression at the DNA level by targeting the first step, ie, factors that affect transcription. Alternatively, the second step may be targeted: if the final product is a protein, translation of mRNA is blocked, and if the final product is RNA, processing or the like is blocked. If the method results in degradation of the RNA ultimately transcribed, the final product is not obtained and thus the second step is inhibited.

  If the disease is caused by gene down-regulation or even by its complete silencing, the therapeutic effect should be an up-regulation of the endogenous gene relative to normal levels. For certain types of cancer, for example, significant tumor suppressor genes are known to be silenced and thus cannot exhibit their corresponding tumor suppressor functions. DNA methylation has been identified as a major reason for gene expression reduction / stopping. Said methylation is often found in the promoter region of the corresponding gene. In these cases, gene expression is restored by removing the methylene group from the DNA. Decreased / stopped expression of a gene also interferes with gene expression by up-regulating a transcription factor that acts as a negative transcription factor for this gene. In these cases, the treatment should be directed to reducing the expression of the gene encoding the transcriptional regulator and thus reducing the level of the transcriptional regulator.

  If up-regulation of the gene is responsible for the disease, the therapeutic effect must be correspondingly reduced expression of the endogenous gene to normal levels. In almost all types of cancer, the gene is known to be up-regulated. These are generally referred to as oncogenes or proto-oncogenes, and proto-oncogenes do not directly affect, for example, cell proliferation, but indirectly by, for example, up-regulating other genes. RNAi methods are used to reduce gene expression. In contrast to demethylating agents that act directly on “DNA-levels”, RNAi methods are typically used in the second step of gene expression, ie, translation into proteins or processing into the corresponding RNA. Works. These methods ultimately lead to degradation of the transcribed RNA. Thus, the entire gene expression process is blocked by RNAi. This is also called “expression silencing” of the gene by RNAi. Mechanistically, artificially introduced “RNA” (eg, transcribed from a vector, introduced as RNA-duplex, etc.) is processed and one strand is transcribed in a sequence-specific manner Embedded in a complex that recognizes Thus, the sequence of the introduced RNA identifies the targeted “gene to be stopped”. After hybridization, recognition, and complex formation, the targeted RNA is degraded by other mechanisms.

  Current methods of monitoring the effects of therapeutic systems that affect gene expression are primarily dependent on comparing disease, eg, tumor status, before and after the therapeutic system is applied. Thus, for example, X-ray or ultrasound methods are used to determine the size of the tumor before and after treatment. However, such a system cannot directly monitor the effect of the treatment on gene expression, nor can it analyze whether the corresponding compound is really localized in the affected tissue. Other methods require a tissue sample to determine the outcome of the treatment. Thus, a biopsy or even a surgical step is necessary, which is often accompanied by additional challenges such as infections. In addition, only post-mortem analysis is possible.

  As a result, there is a need to establish or improve methods that can monitor the effects and applications of these therapies on targeted gene expression and therapies that affect gene expression in a non-invasive manner. .

  It is an object of the present invention to provide compounds and methods for non-invasively monitoring the expression of a gene of interest in a cell when the cell is contacted with a compound that affects the expression of the gene of interest.

  As will become apparent from the following description, these and other objects of the invention are solved by the subject matter of the independent claims. The dependent claims relate to some preferred embodiments of the invention.

According to one aspect of the invention,
a)
aa) a promoter sequence;
bb) a sequence encoding the gene of the polypeptide of interest operably linked to the promoter sequence;
cc) inducing into the cell a nucleic acid molecule having a sequence encoding a fluorescent reporter polypeptide operatively fused to the sequence of bb);
b) detecting the fluorescent reporter polypeptide in the cell by a non-invasive optical imaging method;
c) contacting the cell with a compound that affects the expression of the gene of interest;
d) detecting the fluorescent reporter polypeptide in the cell by a non-invasive optical imaging method during the step c);
e) comparing the level of the fluorescent reporter polypeptide detected in step b) with the level of the fluorescent reporter polypeptide detected in step d);
f) non-invasively monitoring the expression of the gene of interest in the cell comprising at least assigning a change in the expression of the gene of the subject induced by the compound based on the comparison in step e) A method is provided.

In a preferred embodiment relating to the method described above, the present invention provides:
a)
aa) a methylated promoter sequence;
bb) a sequence encoding the gene of the polypeptide of interest operably linked to the promoter sequence;
cc) inducing into the cell a nucleic acid molecule having a sequence encoding a fluorescent reporter polypeptide operatively fused to the sequence of bb);
b) detecting the fluorescent reporter polypeptide in the cell by a non-invasive optical imaging method;
c) contacting the cell with a demethylating agent;
d) detecting the fluorescent reporter polypeptide in the cell by a non-invasive optical imaging method during the step c);
e) comparing the level of the fluorescent reporter polypeptide detected in step b) with the level of the fluorescent reporter polypeptide detected in step d);
f) assigning an increase in the expression of the gene of interest in the subject induced by the demethylating agent based on the comparison in step e) at least, wherein the expression of the gene of interest in the cell is non-invasive in the body Describe how to monitor by sex.

In a more preferred embodiment, the present invention provides
a)
aa) a methylated promoter sequence;
bb) a sequence encoding the gene of the polypeptide of interest operably linked to the promoter sequence;
cc) directing a nucleic acid molecule having a sequence encoding a fluorescent reporter polypeptide operatively fused to the sequence of bb) into a cell outside the human body or animal body;
b) detecting the fluorescent reporter polypeptide in the cell by a non-invasive optical imaging method;
c) contacting the cell with a demethylating agent;
d) detecting the fluorescent reporter polypeptide in the cell by a non-invasive optical imaging method during the step c);
e) comparing the level of the fluorescent reporter polypeptide detected in step b) with the level of the fluorescent reporter polypeptide detected in step d);
and f) assigning an increase in the expression of the gene of the subject induced by the demethylating agent based on the comparison in step e), the method of non-invasively monitoring the expression of the gene of the subject Is described.
Other embodiments of the invention include:
a) a methylated promoter sequence;
b) a sequence encoding the gene of the polypeptide of interest operably linked to the promoter sequence;
c) An isolated nucleic acid molecule that can be used for the above-described method having a sequence encoding a fluorescent reporter polypeptide operatively fused to the sequence of b).

  A coding sequence having a sequence encoding a gene of a polypeptide of interest fused to a sequence encoding a fluorescent reporter polypeptide comprises a single start codon at the 5 'end and a single stop codon at the 3' end. Have The term “single start codon” as used herein does not exclude the presence of other internal ATG codons. However, these latter codons must not initiate translation.

  In a preferred embodiment of the invention, the gene of interest in this aspect of the invention is a suspected gene or tumor suppressor gene that causes disease due to its silencing.

Thus, in another preferred embodiment, the present invention
a) a methylated promoter sequence;
b) CADM1 (SEQ ID No. 2), Rb (SEQ ID No. 1), ZMYND10 (SEQ ID No. 3), RASSF5 (SEQ ID No. 4), PTEN operatively linked to the promoter sequence Encodes a polypeptide selected from the group of polypeptides having (SEQ ID No. 5), SERPINB5 (SEQ ID No. 6), EPB41L3 (SEQ ID No. 7), and DAPK1 (SEQ ID No. 8) An array to
c) an isolated nucleic acid molecule having a sequence encoding a fluorescent reporter polypeptide operably fused to the sequence of b), wherein the coding sequence consisting of b) and c) is 5 It relates to an isolated nucleic acid molecule that can be used for the above-described method with a single start codon at the 'end and a single stop codon at the 3' end.

  Preferably, the sequence referred to in item b) above is selected from CADM1 (SEQ ID No. 2) and / or Rb (SEQ ID No. 1).

In another preferred embodiment, the present invention provides:
a)
aa) a promoter sequence;
bb) a sequence encoding a non-functional polypeptide of the gene of interest operably linked to the promoter sequence;
cc) inducing into the cell a nucleic acid molecule having a sequence encoding a fluorescent reporter polypeptide operatively fused to the sequence of bb);
b) detecting the fluorescent reporter polypeptide in the cell by a non-invasive optical imaging method;
c) contacting the cell with RNAi tuned for the gene of interest;
d) detecting the fluorescent reporter polypeptide in the cell by a non-invasive optical imaging method during the step c);
e) comparing the level of the fluorescent reporter polypeptide detected in step b) with the level of the fluorescent reporter polypeptide detected in step d);
f) assigning a decrease in the expression of the gene in the subject induced by the RNAi based on the comparison in step e) at least, non-invasively reducing the expression of the gene of interest in the cell in vivo Describe how to monitor by sex.

The present invention further provides:
a) a promoter sequence;
b) a sequence encoding a non-functional polypeptide of the gene of interest operably linked to the promoter sequence;
c) a nucleic acid molecule that can be used in such a method, comprising a sequence encoding a fluorescent reporter polypeptide operatively fused to the sequence of b) above.

  A coding sequence having a sequence encoding a nonfunctional polypeptide of a gene of interest fused to a sequence encoding a fluorescent reporter polypeptide has a single start codon at the 5 ′ end and a single stop at the 3 ′ end. Has a codon. The term “single start codon” as used herein does not exclude the presence of other internal ATG codons. However, these latter codons must not initiate translation.

  In a preferred embodiment of this aspect of the invention, the gene of the sequence of interest encoding the non-functional gene of the polypeptide of interest is part of the entire gene of the sequence of interest, or at least one insertion, at least one deletion Any of the genes of the sequence of interest including at least one nucleotide exchange, or a mixture thereof. As a result, a non-functioning gene of the target polypeptide is obtained. However, the gene of interest region does not have a stop codon.

  Furthermore, in a more preferred embodiment of this aspect, the present invention relates to the above two paragraphs before the gene of interest is an oncogene, proto-oncogene, or a gene that is suspected of causing a disease due to overexpression Describe diffusion molecules as outlined.

Thus, in a more preferred embodiment, the present invention provides
a) a promoter sequence;
b) VEGF (SEQ ID No. 10), RAS (SEQ ID No. 11), Wnt (SEQ ID No. 12), MYC (SEQ ID No. 13), ERK operably linked to the promoter sequence. A sequence encoding a non-functional polypeptide selected from the group of polypeptides having (SEQ ID No. 14) and TRK (SEQ ID No. 15);
c) an isolated nucleic acid molecule having a sequence encoding a fluorescent reporter polypeptide operatively fused to the sequence of b), wherein the coding sequence consisting of b) and c) is 5 ′ It relates to an isolated nucleic acid molecule which can be used for the above-mentioned method, having a single start codon at the end and a single stop codon at the 3 ′ end.

  Preferably, the sequence referred to in item b) above is selected from VEGF (SEQ ID No. 10) and / or Wnt (SEQ ID No. 12).

  Thus, in a corresponding preferred embodiment of this aspect of the invention, VEGF (SEQ ID No. 10), RAS (SEQ ID No. 11), Wnt (SEQ ID No. 12), MYC (SEQ ID No. 13) A sequence encoding a non-functional polypeptide selected from the group of polypeptides having ERK (SEQ ID No. 14) and TRK (SEQ ID No. 15) to obtain a non-functional polypeptide, It has either part of the entire sequence, or the entire sequence including at least one insertion, at least one deletion, at least one nucleotide exchange, or a combination thereof. However, the sequence does not have a stop codon.

In other embodiments related to this aspect, the invention provides:
a) a promoter sequence;
b) encoding a non-functional polypeptide of Her-2neu (SEQ ID No. 9) comprising at least one insertion, at least one nucleic acid exchange, or a combination thereof to obtain a non-functional polypeptide; A sequence operatively linked to the promoter sequence and having no stop codon;
c) an isolated nucleic acid molecule having a sequence encoding a fluorescent reporter polypeptide operatively fused to the sequence of b), wherein the coding sequence consisting of b) and c) is 5 ′ It relates to nucleic acid molecules having a single start codon at the end and a single stop codon at the 3 ′ end.

In yet another aspect of the invention,
a)
aa) a methylated promoter sequence;
bb) a sequence encoding the gene of the polypeptide of interest operably linked to the promoter sequence;
cc) directing a nucleic acid molecule having a sequence encoding a fluorescent reporter polypeptide operatively fused to the sequence of bb) into a cell outside the human body or animal body;
b) detecting the fluorescent reporter polypeptide in the cell by a non-invasive optical imaging method;
c) contacting the cell with RNAi tuned for the gene of interest;
d) detecting the fluorescent reporter polypeptide in the cell by a non-invasive optical imaging method during the step c);
e) comparing the level of the fluorescent reporter polypeptide detected in step b) with the level of the fluorescent reporter polypeptide detected in step d);
and f) assigning a decrease in the expression of the gene of the subject induced by the RNAi based on the comparison in step e), wherein the decrease in the expression of the gene of interest is in vitro or in vitro Disclosed is a method that is noninvasively monitored within a cell.

  In embodiments of the in vivo and in vitro methods outlined above, the nucleic acid molecules and / or RNAi are host cells for sonoporation, local injection, or correct targeting, identification and localization or a mixture thereof. It is introduced into the cell by gene transfer with the help of a liposome bag carrying antibodies against the antigen.

  Yet another aspect of the invention relates to the use of the method described above.

  The method described above may be applied outside or inside the human body or outside the animal. One of the above methods is used to monitor the expression of a gene of interest in a cell over time when contacting the cell with a compound that affects the expression of the gene of interest, and thus to monitor the effect of the compound. .

  In another embodiment, the above-described method delivers a compound that affects the expression of a gene of interest in a cell and not only monitors the delivery of the compound but also simultaneously expresses the gene of the subject induced by the compound. Used to monitor the impact on. This is also provided for in vivo situations as well as methods performed on cells outside the human body or against animals.

  In another preferred embodiment, the above-described method comprises administering a dose of a compound that affects the expression of a gene of interest in order to obtain further changes in the expression of the gene of interest, either inside or outside the human body or animal. Used to adjust.

  In another aspect of the present invention, methods relating to the use of demethylating agents can test different demethylating agents in one embodiment in order to identify the most potent demethylating agents separately or in combination. Used to do.

  In a further preferred embodiment, this aspect relates to a method comprising RNAi. Thus, the method is used to test different RNAi that are tailored to one gene of interest in order to identify the most potent RNAi separately or in combination.

FIG. 1 illustrates the schematic and exemplary steps of a possible application of the present invention, namely RNAi therapy and its monitoring in breast cancer tissue.

  These steps are described in detail in the exemplary section of the application.

  The inventors have found that the expression of a gene of interest in a cell can be monitored non-invasively. This system is used when contacting cells with compounds that affect the expression of the gene of interest.

  While exemplary embodiments of the present invention are described in detail, important definitions are provided for understanding the present invention.

  As used in the specification and appended claims, the singular forms “a” and “an” also include the plural unless the context clearly dictates otherwise.

  In the text of the present invention, the terms “about” and “approximately” indicate intervals of accuracy understood by those skilled in the art to still ensure the technical effect of the feature. The term typically indicates a deviation of ± 10%, preferably ± 5% of the indicated numerical value.

  The term “having” should be understood as not limiting. For the purposes of the present invention, the term “consisting of” is considered to be a preferred embodiment of the term “comprising of”. From now on, if a group having at least a certain number of embodiments is defined, this also means that it preferably includes a group consisting only of these embodiments.

As described above, the present invention describes a method for non-invasively monitoring the expression of a gene of interest when contacting the cell with a compound that affects the expression of the gene of interest. Thus, in one embodiment, the present invention is a non-invasive method for monitoring the expression of a gene of interest in a cell comprising:
a)
aa) a promoter sequence;
bb) a sequence encoding the gene of the polypeptide of interest operably linked to the promoter sequence;
cc) inducing into the cell a nucleic acid molecule having a sequence encoding a fluorescent reporter polypeptide operatively fused to the sequence of bb);
b) detecting the fluorescent reporter polypeptide in the cell by a non-invasive optical imaging method;
c) contacting the cell with a compound that affects the expression of the gene of interest;
d) detecting the fluorescent reporter polypeptide in the cell by a non-invasive optical imaging method during the step c);
e) comparing the level of the fluorescent reporter polypeptide detected in step b) with the level of the fluorescent reporter polypeptide detected in step d);
and f) assigning a change in the expression of the gene in the subject induced by the compound based on the comparison in step e).

  In the context of the present invention, the term “non-invasively” monitoring means direct physical contact with the patient's cells and / or monitored tissue during the detection steps b) and d) of the inventive method. Means that it is not necessary to monitor the expression of the gene of interest. Thus, there is no need for surgery, biopsy, or other methods (such as patch clamping for in vitro analysis) involving contact with cells during the detection step.

  “Monitoring”, in a general sense, refers to the process of determining parameters and keeping track of parameters over time.

  As used herein, the term “expression of a gene of interest” has at least two steps. Transcription of the gene of interest into the corresponding RNA is one step in gene expression. When a protein is encoded by a gene, RNA is called mRNA. The RNA is further processed (eg, conjugated) and in a second step the mRNA is translated into a protein. Thus, the protein is the final product of gene expression. The amount of protein as the final product correlates with the level of gene expression. However, the RNA molecule may also be the end product of gene expression. The RNA may also follow the processing and modification of the second step.

  The term “compound that affects gene expression” as used herein includes compounds that have a positive or negative effect on endogenous gene expression.

  Such compounds operate at the DNA level. This means that the compound leads to a structural rearrangement of the DNA, for example from a closed structure to an open structure, without affecting the methylation status of the DNA or affecting the DNA sequence, for example. Since DNA methylation (especially in the promoter region) is known to cause silencing of the corresponding gene, a compound acting as a demethylating agent removes the methylation group from the DNA, thus increasing gene expression It has been known. Histone deacetylase (HDAC) is involved in gene suppression by removing acetyl groups from DNA. Thus, compounds that inhibit HDAC have a positive effect on gene expression as well. These drugs, commonly referred to as HDAC inhibitors, will also be described below. However, one embodiment of the invention includes compounds that positively affect gene expression.

  In another preferred embodiment of the invention, a compound that acts on the second step of gene expression, ie transcribed RNA, is used. When the RNA is degraded, gene expression is negatively affected. In this embodiment of the invention, for example, RNAi is used to inhibit the translation step. Such RNAi includes siRNA, shRNA, miRNA, etc., as described in detail below.

  As used herein, “contacting a cell” has any aspect known to those skilled in the art to provide a compound that can exert an effect on the cell. This may be done in vivo by, for example, intravenous infusion, local administration, inhalation and the like. For cell culture systems, compounds such as demethylating agents may simply be added to the cell culture medium used for culture. However, gene transfer methods such as those outlined below may also be used to bring a compound into contact with a cell, which may be introduced into the cell in the next step.

  The term “nucleic acid molecule” in the context of the present invention refers to DNA, RNA or derivatives thereof. Preferably, a nucleic acid molecule refers to a double-stranded DNA molecule. Depending on the mechanism used to introduce the nucleic acid molecule into the cell, the nucleic acid molecule is circular or linear.

  In the context of the present invention, the term “introducing into a cell” defines that a nucleic acid molecule, such as DNA or RNA, is introduced into the corresponding cell by methods known to those skilled in the art. Furthermore, the term “introducing” means that the nucleic acid molecule is present in the cell such that a coding sequence present in the nucleic acid molecule is expressed depending on what region is adjusted. For this purpose, techniques such as gene transfer methods as described in detail below are used.

  In the context of the present invention, the term “coding region” or “coding sequence” is meant to encompass a DNA sequence starting at the 5 ′ end with an initiation codon, ie, ATG. When translation of mRNA is initiated, this triplet encodes for the first amino acid of the corresponding protein, methionine. Thus, the coding region ends at the 3 ′ end with a stop codon, eg TAG, to finish protein translation. It is important for the present invention to note that there are no stop codons anywhere in the coding sequence other than those described above at the three ends. Thus, in summary, the entire coding sequence is transcribed into one corresponding mRNA. Translation of mRNA results in one protein consisting of at least two different domains / polypeptides / parts. However, as pointed out in detail below, the polypeptide need not necessarily be functional, but for every coding sequence, at least two sequence encodings for at least two polypeptides are: Present within the one coding sequence.

  In a preferred embodiment of the invention, the coding sequence of the nucleic acid molecule has a single start codon at the 5 ′ end and a single stop codon at the 3 ′ end. The term “single start codon” as used herein means that the sequence may have other internal ATG codons, but this is not used to initiate translation.

  The terms "gene of interest sequence", "region of interest gene" and "sequence encoding said gene of interest polypeptide" refer to the DNA sequence of the gene of interest to be monitored by the method of the invention. . In the context of the present invention, genes that are translated into proteins and are suspected of causing certain diseases due to misregulation are preferably encompassed by the term. This is outlined in detail below. Thus, such sequences have only the coding region of the gene, ie, only exons, and no introns. However, in certain embodiments, it is also applicable to use the intron region of the gene of interest.

  In the following, the term “reporter polypeptide” as well as the term “reporter” will be explained in detail. This paragraph refers to all the methods described in detail below, as well as all examples of the present invention, ie, all nucleic acid molecules encompassed by the present invention. The sequence encoding the reporter polypeptide is in frame with a unique ATG at the 5 ′ end of the coding sequence so that the corresponding polypeptide is always correctly expressed, ie a functional polypeptide / protein is made. There is always. Furthermore, in all embodiments of the invention, the nucleic acid sequence encoding the reporter polypeptide encodes a fluorescent reporter polypeptide. The fluorescent reporter polypeptide is selected from blue, green, cyan, red or infrared fluorescent protein. In a particularly preferred embodiment of the invention, the reporter sequence encodes a green fluorescent protein.

  As used herein, the term “operably fused” refers to at least two DNA regions (eg, a sequence encoding a gene of a polypeptide of interest as well as a sequence encoding a reporter polypeptide). Define that they are linked to each other. Thus, in one preferred embodiment, they form one expression product consisting of at least two different domains / polypeptides, ie one coding sequence for one protein. Thus, as described above, in order to express a functional reporter polypeptide, it is only necessary to code for the corresponding protein whose sequence encoding the reporter polypeptide is always in frame with the translation region. There are two translation domains. Preferably, the gene of interest sequence is fused 5 'to the reporter sequence to encode a protein made of the gene of interest polypeptide followed by a fluorescent reporter polypeptide.

  In a further preferred embodiment of the invention, the coding region of such a nucleic acid molecule additionally has a spacer region encoding a polypeptide spacer between the fused gene of the subject and the reporter sequence. Also in this case, the spacer region is always in frame with the translation region in order to express a functional space polypeptide.

  The term “operably linked” means that the expression of the coding region defined above is mechanically controlled by other regions such as promoters or enhancers. These regions are usually other DNA regions that are also present in the nucleic acid molecule. Such operably linked regions may be anywhere in the nucleic acid molecule, but they are preferably present as regulatory regions such as 5 promoters in the coding sequence.

  In the context of controlling the expression of a coding sequence, the term “promoter sequence” or simply “promoter” refers to a defined DNA sequence that affects the expression of a gene. Thus, a typical promoter sequence consists of a defined base sequence of a particular length that is recognized by a particular cellular factor. Often, these factors bind to these conserved sequences and act as positive or negative regulators of gene expression and are therefore commonly referred to as “transcriptional regulators”. Many promoter sequences are known to those skilled in the art, and even combinations of different promoter sequences of artificial nucleic acid molecules are possible.

  Depending on the method of the invention, different promoters may be used. Thus, a nucleic acid molecule according to the present invention has, in a preferred embodiment, a promoter sequence in which the promoter sequence is a human promoter identified by at least one human transcriptional regulator that induces the expression of the coding sequence. Furthermore, the human promoter is a strong and constitutively active promoter. In another embodiment, the human promoter is a strongly inducible, eg, tissue selective promoter. In a particular embodiment, the human promoter may be a viral promoter. In the most preferred embodiment of the present invention, the promoter sequence of the nucleic acid molecule that regulates the expression of a coding sequence having a gene of interest sequence in particular is an endogenous promoter sequence (ie, a sequence that regulates expression of an endogenous gene of interest). Therefore, it reflects the endogenous situation.

  The term “method of detecting by non-invasive optical imaging” refers to a method used to non-invasively detect a fluorescent reporter polypeptide in a cell. Since the method relies on a reporter polypeptide, insights can be gained not only on the cells in which the polypeptide is expressed, but also on the functional and biochemical properties of the polypeptide. The “optical imaging” method usually relies on a drug, commonly referred to as a contrast agent, and in the present invention, a fluorescent reporter polypeptide represents the contrast agent.

  Preferably, detection of a fluorescent reporter polypeptide by the method of the present invention is done by fluorescence microscopy, fluorescence imaging, single and multi-photon confocal microscopy, laser induced fluorescence, and the like. Since fluorescent proteins emit a specific wavelength of color upon excitation, intracellular fluorescent proteins are excited by using light of a specific excitation wavelength followed by detection of the emitted wavelength of light. (For example, GFP is excited by ultraviolet rays and emits light having a wavelength corresponding to green). Thus, the imaging method used in the present invention emits light at a specific wavelength to excite a fluorescent polypeptide, and detects light of a specific wavelength emitted by the fluorescent polypeptide. Refer to the device. Of course, the device is coupled to a conventional optical microscope, monitor, data storage device, and the like. The device is arranged according to the area to be monitored. Said area consists for example of a single cell, a cell population of a specific area of a cell culture dish, or a human or animal cell, preferably several cells of a human or animal monitored by the method of the invention It may be an organization. Any device capable of detecting fluorescent polypeptides known to those skilled in the art may be used.

  In step b) of the method of the invention, the level of the fluorescent reporter polypeptide is detected before the cell is contacted with a compound that affects the expression of the gene of interest. Thereafter, in a subsequent step, the reporter polypeptide is again detected after contact with the compound affecting the expression of the gene of interest. In this way, two different levels of reporter polypeptide are detected. It is then possible to “compare” these two levels as outlined in step e). However, the present invention is not intended to rely solely on the two detection steps. In other preferred embodiments of the invention, the second detection step can be repeated at least 2, 3, 4, 5, 6, 10 or 100 times to obtain data over time. Thus, in one embodiment, the present invention relates to a method wherein the second detection step d) is performed several times to determine the change in the expression of the gene of interest over time. Thus, in the above example, it is possible to analyze the level of reporter polypeptide over time.

  In another preferred embodiment, it is also possible to record the signal in real time in order to obtain a readout, ie a moving picture of the detection of the fluorescent protein. This is called “live cell imaging” or “live tissue imaging”.

  Of course, the time points are adjusted according to the compound that affects the expression of the gene of interest. For example, if the compound is known to slowly act on the expression of the gene of interest, the level of the fluorescent reporter polypeptide is increased after an additional time of, for example, 5, 10, 20, 30 or 60 minutes from the time of introduction of the compound. It would be useful to detect. If a fast acting compound is used, the second detection step is performed within seconds depending on the compound introduction.

  Furthermore, the term “compare” should be understood as comparing the relative amount of reporter polypeptide in at least two detection steps. In one preferred embodiment, the present invention describes the method outlined above wherein the comparison step e) is by analyzing the relative amount of reporter polypeptide detected in steps b) and d). To do. Thus, it is not necessary to quantify the absolute amount of the polypeptide. However, when using such a relative quantity, it is guaranteed that the quantity is normalized to a value or control that is the same in at least two detection steps in order to obtain a normalized relative quantity. It is necessary to

  The term “assign change” in the context of the present invention means that after said comparison, the difference between at least two detection steps is expressed as a percentage, for example. This includes a percentage decrease or percentage increase in the reporter polypeptide over a specific detection time. When detecting at least two reporter polypeptides, it may be concluded that the level of reporter polypeptide did not change between the two time points or over time.

  The method of the present invention may be used in vivo or in vitro.

  In the “in vivo situation”, the cells described in the method of the present invention may be part of human or animal tissue, or may be cells circulating in the human or animal body. In this situation, not only a single cell, but preferably a cell known or suspected to cause a specific disease or a cell of a specific tissue is the target of the present invention. As outlined above, misregulation of certain important cell regulators (eg, cell cycle regulators such as pRB, cyclin, CdK (cyclin dependent kinase), etc.) leads to diseases such as cancer. In an in vivo setup, the method according to the invention is used to monitor the expression of a gene of interest in, for example, a cancer tissue or tumor, when a compound that affects expression is administered. A preferred embodiment of this in vivo situation according to the present invention will be described in detail below.

Thus, in a preferred embodiment, the present invention is a non-invasive method for monitoring in vivo expression of a gene of interest in a cell comprising:
a)
aa) a promoter sequence;
bb) a sequence encoding the gene of the polypeptide of interest operably linked to the promoter sequence;
cc) inducing into the cell a nucleic acid molecule having a sequence encoding a fluorescent reporter polypeptide operatively fused to the sequence of bb);
b) detecting the fluorescent reporter polypeptide in the cell by a non-invasive optical imaging method;
c) contacting the cell with a compound that affects the expression of the gene of interest;
d) detecting the fluorescent reporter polypeptide in the cell by a non-invasive optical imaging method during the step c);
e) comparing the level of the fluorescent reporter polypeptide detected in step b) with the level of the fluorescent reporter polypeptide detected in step d);
and f) assigning a change in the expression of the gene in the subject induced by the compound based on the comparison in step e).

  In a preferred embodiment of this in vivo setting, the present invention provides that the nucleic acid molecule and / or the RNAi host sonoporation, localized injection, or antibody for correct targeting, identification and localization or a combination thereof. We describe a method as outlined above that is introduced into the cell by gene transfer with the help of a liposome bag that delivers it to the cell antigen. Such techniques are known to those skilled in the art.

  Furthermore, the present invention provides that in preferred in vivo embodiments, the fluorescent reporter polypeptide encoded by the reporter sequence is selected depending on the depth of the tissue to be monitored, i.e. against superficial tissue. Reference is made to a method wherein the fluorescent protein is selected from blue, green or cyan fluorescent proteins and, for deep tissue, selected from red or infrared fluorescent proteins.

  The method of the present invention may be used ex vivo. Such an in vitro setup is preferably a mammalian cell cultured in a cell / tissue culture system, or extracted from the human or animal body and used and / or processed outside the human or animal body. Refers to a system that uses cells.

  A “mammalian cell culture system” according to the present invention includes all cell lines known and used by those skilled in the art. These cell lines may be human, mouse, rat, hamster or chicken cell lines and may be cultured by standard techniques. Of course, different human cell systems may be used, such as healer cells, 293 cells, WI-38 cells, U2OS cells, and the like.

Thus, one embodiment of the present invention is
a)
aa) a methylated promoter sequence;
bb) a sequence encoding the gene of the polypeptide of interest operably linked to the promoter sequence;
cc) directing a nucleic acid molecule having a sequence encoding a fluorescent reporter polypeptide operatively fused to the sequence of bb) into a cell outside the human body or animal body;
b) detecting the fluorescent reporter polypeptide in the cell by a non-invasive optical imaging method;
c) contacting the cell with a compound that affects the expression of the gene of interest;
d) detecting the fluorescent reporter polypeptide in the cell by a non-invasive optical imaging method during the step c);
e) comparing the level of the fluorescent reporter polypeptide detected in step b) with the level of the fluorescent reporter polypeptide detected in step d);
f) non-invasively monitoring the expression of the gene of interest in the cell comprising at least assigning a change in the expression of the gene of interest of the subject induced by the compound based on the comparison in step e) Have a method to do.

  In a preferred embodiment of this in vitro setting, the present invention provides that the nucleic acid molecule and / or the RNAi host sonoporation, localized injection, or antibody for correct targeting, identification and localization or a mixture thereof. We describe a method as outlined above that is introduced into the cell by gene transfer with the help of a liposome bag that delivers it to the cell antigen. Such techniques are known to those skilled in the art.

  The change in the expression of the gene of interest monitored by the method according to the invention may be an up-regulation or down-regulation of said gene of interest. As described above, this is analyzed in vivo or in vitro. In the first situation or disease, the gene of interest is down-regulated, so the goal is up-regulation of the gene of interest and monitoring of the up-regulation. The second situation is that the gene of interest is upregulated in the disease. In such a situation, the goal is to down-regulate the corresponding gene and monitor the down-regulation by the method of the present invention. Preferred embodiments of the nucleic acid molecule as well as the method according to the invention for each situation are described below. These examples are intended to illustrate the situation. Thus, other compounds with the same overall effect of either down-regulation or up-regulation of gene expression may be used.

  Where the gene of interest is downregulated, in a preferred embodiment, the method of the invention involves the use of a demethylating agent to upregulate such a gene. In the reverse situation, when down-regulation of the gene of interest is the goal, the method of the invention refers in a preferred embodiment to the use of RNAi. Now these two groups of preferred embodiments will be described in more detail.

  In order to analyze whether a gene has been down-regulated / silenced for DNA methylation, a differential methylation study is made prior to utilizing the monitoring method of the present invention prior to determining treatment. This is done using fine needle aspiration cytology. DNA samples are obtained using this fine needle aspiration technique and analyzed for these methyl groups. Such methods are known to those skilled in the art.

  In the following paragraphs, preferred methods of monitoring the increase in the expression of the gene of interest and preferred embodiments of the invention referring to nucleic acid molecules are described.

Thus, in one embodiment, the present invention provides
a) a methylated promoter sequence;
b) a sequence encoding the gene of the polypeptide of interest operably linked to the promoter sequence;
c) An isolated nucleic acid molecule having a sequence encoding a fluorescent reporter polypeptide operatively fused to the sequence of bb).

  The coding sequence consisting of b) and c) has a single start codon at the 5 'end and a single stop codon at the 3' end. The term “single start codon” as used herein means that the sequence may have other internal ATG codons, but is not used to initiate translation.

  As already mentioned above, it is known that methylation of DNA, particularly promoters, inhibits the expression of genes controlled by the promoters. DNA methylation is preferably understood as base methylation, preferably as cytosine methylation. Genes that are operably linked to methylated promoter sequences are not described and are therefore not active. For the nucleic acid molecule, if the promoter sequence remains methylated, the corresponding coding sequence consisting of b) and c) will not be expressed. However, if the region is no longer modified by a methyl group, the sequence is a human promoter sequence identified by at least one human transcriptional regulator that induces expression of the coding region. Thus, demethylation of the methylated and promoted region leads to an unmethylated region that can drive expression of genes that are operably linked to the region. The present invention, in one embodiment, is a human promoter wherein, upon action of a demethylating agent, the unmethylated promoter sequence is identified by at least one human transcriptional regulatory factor that induces expression of the coding sequence. It has a nucleic acid molecule as just described. In a preferred embodiment, the methylated promoter of the nucleic acid molecule is identical to the endogenous methylation promoter of the gene of interest.

  In a preferred embodiment of the present invention, the claimed nucleic acid molecule as described in the paragraph above is a sequence of the subject whose gene of interest is a tumor suppressor gene or a gene suspected of causing a disease due to silencing. Have a gene. In one example of such an embodiment, the gene of interest is RB. In other cases, the gene of interest is CADM1, ZMYND10, RASSF5, PTEN, SERPINB5, EPB41L3, DAPK1, or the like.

  Said composition / nucleic acid molecule is in another embodiment used in either an in vivo method or an in vitro method according to the present invention.

  The term “demethylating drug” or “demethylated compound” as used herein is meant to encompass any drug capable of DNA demethylation, ie, capable of removing a methyl group from intracellular DNA, preferably from cytosine. . The compound is selected from the group of compounds having 5-azacytidine and the like. The systematic IUPAC name for 5-azacytidine is 4-amino-1- [3,4-dihydroxy-5- (hydroxymethyl) oxolan-2-yl] -1,3,5-triazine-2-1. Chemical analogues of cytidine are incorporated into DNA and RNA during DNA replication and DNA transcription, respectively. This integration inhibits the activity of methyltransferase enzymes such as DNMT1. Thus, integration results in demethylation. Of course, those skilled in the art are also known as other cytidine-analogs and / or 5-aza-2'deoxytidine, 1- (β-D-ribofuranosyl), dehydro-5-aza-cytidine, Zebularine [ We know compounds that act like 5-azacytidine, such as 1- (β-D-ribofuranosyl) -1,2-dihydropyrimidine-2-1]. Their mechanism of action is based on inhibiting methyltransferases by forming complexes and / or reducing methyltransferases, etc. As already mentioned above, the effect of such drugs on methylated promoter regions is the activation of regulatory genes.

Thus, in a preferred embodiment, the present invention
a)
aa) a methylated promoter sequence;
bb) a sequence encoding the gene of the polypeptide of interest operably linked to the promoter sequence;
cc) directing into the cell a nucleic acid molecule having a sequence encoding a fluorescent reporter polypeptide operatively fused to the sequence of bb);
b) detecting the fluorescent reporter polypeptide in the cell by a non-invasive optical imaging method;
c) contacting the cell with a demethylating agent;
d) detecting the fluorescent reporter polypeptide in the cell by a non-invasive optical imaging method during the step c);
e) comparing the level of the fluorescent reporter polypeptide detected in step b) with the level of the fluorescent reporter polypeptide detected in step d);
f) assigning an increase in the expression of the gene of interest in the subject induced by the demethylating agent based on the comparison in step e) at least comprising in vivo expression of the gene of interest in the cell Relates to non-invasive monitoring methods.

In another preferred embodiment, the present invention provides:
a)
aa) a methylated promoter sequence;
bb) a sequence encoding the gene of the polypeptide of interest operably linked to the promoter sequence;
cc) directing a nucleic acid molecule having a sequence encoding a fluorescent reporter polypeptide operatively fused to the sequence of bb) into a cell outside the human body or animal body;
b) detecting the fluorescent reporter polypeptide in the cell by a non-invasive optical imaging method;
c) contacting the cell with a demethylating agent;
d) detecting the fluorescent reporter polypeptide in the cell by a non-invasive optical imaging method during the step c);
e) comparing the level of the fluorescent reporter polypeptide detected in step b) with the level of the fluorescent reporter polypeptide detected in step d);
and f) assigning an increase in the expression of the gene in the subject induced by the demethylating drug based on the comparison in step e).

  In in vivo and in vitro situations, the present invention describes in a preferred embodiment a method in which the amount of demethylating drug is increased or decreased by the allocation of step f) listed above. Furthermore, the present invention provides that in a preferred embodiment, different demethylating agents are used separately or in combination to obtain the most potent increase in the expression of the gene of interest by assignment in step f) listed above. Describes how it will be tested.

  The down-regulation of the gene of interest is also due to the conformational restriction of the corresponding DNA sequence. In particular, it is known that the closed conformation of the promoter region negatively affects gene expression. The “closed” or “packed” DNA conformation appears to correlate with the acetylation state of the DNA. Thus, acetyl groups on DNA result in a more open DNA conformation, and thus apparently affect gene expression. Such acetyl groups are transferred to DNA via histone-acetyltransferase (HAT) and removed from the DNA by histone-deacetylase (HDAC). By inhibiting HDAC, the acetylation status of DNA is increased, thus up-regulating gene expression. Typical HDAC inhibitors used in the methods of the invention are 3- (1-methyl-4-phenylacetyl-1H-2-pyrrolyl) -N-hydroxy-2-propenamide, cyclo [ (2S) -2-amino-8-oxo-decanoyl-1-methoxy-L-tryptophanyl-L-isoleucyl- (2R) -2-piperidineexcarbonyl (sodium butyrate), 4 , 5: 8,9-Dianhydro-1,2,6,7,11-pentadeoxy-D-threo-D-ido-undeca-1 , 6-Dienitol tol), 6- (1,3-Dioxo-1H, 3H-benzo [de] iso-quinolin-2-yl) -hexanoic acid hydroxy amide, 2- [amine (2-hydroxynaphthalene) -L-ylmethylene)]-N- (1-phenethyl) benzamide, [R- (E, E)]-7- [4- (dimethylamino) phenyl] -N-hydroxy-4,6-dimethyl -7-oxo-2,4-heptadienamide and the like. These inhibitors are supplied, for example, by SigmaAldrich (see http://www.sigmaaldrich.com/Area_of_Interest/Life_Science/Cell_Signaling/Product_Highlights/Histone_Deacetylase_Inhibitors.html). Other examples of HDAC inhibitors are PXD101 (also called Belinostat) and SAHA. In particular in this embodiment of the invention, the promoter region of the nucleic acid molecule used in the method of the invention corresponds to the endogenous promoter of the gene of interest and thus reflects the DNA conformation of the endogenous promoter.

Thus, in a preferred embodiment, the present invention is a non-invasive method for monitoring in vivo expression of a gene of interest in a cell comprising:
a)
aa) a promoter sequence;
bb) a sequence encoding the gene of the polypeptide of interest operably linked to the promoter sequence;
cc) inducing into the cell a nucleic acid molecule having a sequence encoding a fluorescent reporter polypeptide operatively fused to the sequence of bb);
b) detecting the fluorescent reporter polypeptide in the cell by a non-invasive optical imaging method;
c) contacting the cell with an HDAC inhibitor;
d) detecting the fluorescent reporter polypeptide in the cell by a non-invasive optical imaging method during the step c);
e) comparing the level of the fluorescent reporter polypeptide detected in step b) with the level of the fluorescent reporter polypeptide detected in step d);
and f) assigning an increase in the expression of the gene in the subject induced by the HDAC inhibitor based on the comparison in step e).

In another preferred embodiment, the present invention provides:
a)
aa) a promoter sequence;
bb) a sequence encoding the gene of the polypeptide of interest operably linked to the promoter sequence;
cc) directing a nucleic acid molecule having a sequence encoding a fluorescent reporter polypeptide operably fused to the sequence of bb) into a cell outside the human body or outside the animal body;
b) detecting the fluorescent reporter polypeptide in the cell by a non-invasive optical imaging method;
c) contacting the cell with an HDAC inhibitor;
d) detecting the fluorescent reporter polypeptide in the cell by a non-invasive optical imaging method during the step c);
e) comparing the level of the fluorescent reporter polypeptide detected in step b) with the level of the fluorescent reporter polypeptide detected in step d);
and f) assigning an increase in the expression of the gene in the subject induced by the HDAC inhibitor based on the comparison in step e).

  In the following paragraphs, preferred embodiments of the invention will be described that refer to methods and nucleic acid molecules for monitoring the decrease in expression of a gene of interest.

Thus, in one embodiment, the present invention provides
a) a promoter sequence;
b) a sequence encoding a non-functional polypeptide of the gene of interest operably linked to the promoter sequence;
c) a sequence encoding a fluorescent reporter polypeptide operatively fused to the sequence of b), wherein the coding region consisting of b) and c) has an initiation codon at at least one 5 ′ end. A nucleic acid molecule of the in vivo method of the present invention having a single stop codon at the 3 ′ end is described.

  In a preferred embodiment of the invention, the claimed nucleic acid molecule as described in the above paragraph comprises a gene of interest suspected of causing a disease due to an oncogene, proto-oncogene, or overexpression Have the gene of interest sequence. In one example of such an embodiment, the gene of interest is Her-2neu. In other examples, the gene of interest is VEGF, RAS, Wnt, MYC, ERK, TRK, and the like.

  With reference to these preferred embodiments of the invention, it is clear why the gene of interest polypeptide is preferably not functional in an in vivo setup. If functional, this will lead to a further increased overall expression of the gene (often an oncogene) resulting from the overexpression of the exogenous gene and the endogenous gene of the sequence of interest. . This is detrimental to cells / tissues.

  Thus, in a preferred embodiment relating to the nucleic acid molecules of the invention and in vivo methods, the gene of the sequence of interest encoding a non-functional polypeptide is obtained in order to obtain a non-functional gene of the polypeptide of interest. It has either part of the entire gene of the sequence of interest, or any of the entire genes of the sequence of interest including at least one insertion, at least one deletion, at least one nucleotide exchange or a mixture thereof. However, this does not have a stop codon. Thus, for example, a frameshift resulting from at least one insertion or at least one deletion in a gene of interest sequence results in a non-functional gene of the subject polypeptide. This is also obtained by a single nucleotide exchange that results in amino acid substitution of the corresponding polypeptide, leading to the destruction of the resulting three-dimensional structure of the protein. If the oncogene is the gene of interest, remove the sequence that encodes the functional part of the resulting oncogene (such as the catalytic region, binding region, dimerization region, etc.) and replace the remaining coding sequence It may be used as a gene of interest sequence of the nucleic acid molecule of the present invention. In this case, the resulting polypeptide is not functional. Of course, those skilled in the art know that multiple sequences are removed and the length of the removed sequence varies depending on the gene of interest.

  When the gene of interest encodes a transcriptional regulatory factor, the promoter sequence of the nucleic acid molecule as described in this aspect of the invention is, in other embodiments, the human transcriptional control encoded by the gene of interest. It is a human promoter that is recognized only by factors. Thus, if down-regulation of the gene of interest is achieved, the expression of the coding sequence (and thus the fluorescent reporter polypeptide) is also due to the reduced level of the gene of interest polypeptide that is the corresponding transcriptional regulator. Controlled downward.

  Said construct / nucleic acid molecule is used in another embodiment either in an in vivo method or an in vitro method according to the present invention.

  In this context, the present invention describes a method for monitoring a decrease in the expression of a gene of interest. Said reduction is achieved by compounds / molecules having antisense RNA, shRNA, siRNA, miRNA or other nucleic acid molecules of equivalent function commonly referred to as “RNAi”, “RNA interference” or “inhibitor molecule”. In addition, the molecules are modified by nanoparticles, for example.

  Using such RNA interference strategies, it is important to use molecules that specifically interfere only with the expression of the gene of interest, and not with other unrelated cellular factors. The selection, identification and production of such RNA-based inhibitors are well known to those skilled in the art.

  Typically, for example, in view of the lack of a second component that is inhibitory to in vivo interactions between the suppressor RNA-based molecule and the respective mRNA, an antisense strategy or siRNA-based Identify sequences within the coding sequence of the gene of interest that are accessible to the technique. Such extension of the nucleic acid is identified using a computer program such as SFOLD.

  In the second step, such sequences are then compared to other sequences to determine if they are unique for each gene of interest. This is done, for example, by performing a BLAST search at the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/).

  Having an identified unique sequence, an antisense or siRNA inhibitor is selected by using a complementary sequence that preferably exactly matches the identified sequence. Of course, a low degree of complementarity may be used if complementarity does not decrease to a level where an unclear interaction between the antisense or siRNA inhibitor and the messenger RNA of other cellular factors can occur.

  Typically, the degree of interpolation between a suppressor RNA-based molecule and a particular sequence identified is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, At least 96%, at least 97%, at least 98% or at least 99%.

  The term “complementary” refers to the basic properties of nucleic acid molecules that hybridize to each other in view of the ability of guanidine to pair with cytidine and adenosine to pair with thymine and uracil. Well known to those skilled in the art.

  Those skilled in the art will, of course, ensure that the nucleic acid sequence that provides complementarity to the complete coding sequence of the gene of interest or a portion thereof is actually unique to the gene of interest. Understand that you must have a certain minimum length.

  Accordingly, the nucleic acid sequence within the suppressor molecule that provides complementarity with the gene of interest is at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 50, at least 75, at least 100, at least 150, at least 200, at least 300 or At least 400 nucleotides in length and should form adjacent extensions.

  A person skilled in the art may specifically target only the gene of interest the nucleic acid sequence to be complementarily applied, depending on the length of the nucleic acid sequence that is complementary to the coding sequence of the gene of interest or part thereof. You will also notice that the minimum degree of complementarity required to ensure that will change. Thus, if a rather long nucleic acid molecule of 30, 50, 60, 75, 100 nucleotides is used, a low degree of complementarity may be used, or the suppressor molecule and the coding sequence of the gene of interest or part thereof Of course, when the complementary conferring nucleic acid sequence in the suppressor molecule only has an extension of, for example, at least 14, 15, 16, 17 or 19 nucleotides, of course, a high degree of complementation. Sexuality will be required.

  As mentioned above, a reduction in the expression of a gene of interest is achieved by “RNAi”, “RNA interference” or “inhibitor molecule” designed as outlined above.

  Thus, one class of such inhibitor molecules are molecules having recombinant nucleic acid molecules with nucleic acid sequences that are complementary to the complete coding sequence of the gene of interest or a portion thereof. Nucleic acid sequence molecules may be made of DNA, RNA or other nucleic acid-based molecules, but instead of nucleotides as long as the resulting molecule can specifically hybridize with the complete coding sequence of the gene of interest or part thereof You may have an analog at least partially.

  In preferred embodiments, the suppressor molecules described above preferably have antisense RNA, shRNA, siRNA, miRNA, or other nucleic acid molecules that function equivalently to the gene of interest.

  These terms clearly indicate to those skilled in the art how these molecules are synthesized, how much complementarity should be considered, etc. It should be understood that antisense RNA as well as shRNA and siRNA according to the present invention meet the above criteria for the length, degree of complementation, etc. of these nucleic acid molecules.

  Antisense molecules are typically 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, It has a length of 30, 50, 70, 100, 150, 200, 300 or more nucleotides.

  For RNA interference purposes, so-called short hairpin RNAs or synthetic duplex siRNA oligonucleotides may be used.

  siRNA molecules typically have a length of 20, 21, 22, 23, 24 or 25 nucleotides.

  Typically, since shRNA molecules are expressed, single stranded RNA molecules form an intramolecular hairpin structure. SiRNA molecules are generated therefrom by intracellular treatment through the enzyme Dicer.

  Intracellular transcription of the shRNA molecule can be achieved if both strands of the shRNA duplex are expressed using an expression vector in the form of a single RNA or DNA molecule. For this purpose, the transcribed RNA strand ideally has 19 to 21 nucleotides of the sense shRNA sequence and ideally 19 to 21 nucleotides of the corresponding complementary sequence. Both sequences are ideally separated, for example by a 6 nucleotide long spacer.

  It is well known that the efficiency of siRNA-mediated suppression of cellular target transcription depends on the selection of appropriate siRNA sequences. Guidelines have been developed for the design of effective siRNA molecules. These guidelines are typically derived for synthetic siRNA oligonucleotides, but should also apply to the processing format of shRNA molecules.

  In addition to shRNA molecules obtained only after intracellular expression and processing, synthetic siRNA oligonucleotides typically consist of double-stranded RNA molecules between 19 and 21 nucleotides in length. These siRNA molecules are, for example, transferred into a cell line as described above, thus initiating RNAi treatment.

  The determination of the siRNA sequence motif and target sequence is determined according to well-known publications such as, for example, the publication by Tuschl et al. Therefore, the coding region of the target mRNA is used to identify only the appropriate siRNA target sequence. While it is preferred that the selected siRNA sequence motif is directed to the coding region of the target mRNA, it may also be designed for regulatory regions of the gene of interest, such as the 5 'and 3' untranslated regions.

  When a messenger RNA coding sequence is used as the target sequence, one typically uses a sequence that begins with a nucleotide 70 downstream of the start codon and ends with a nucleotide 50 upstream of the stop codon.

  This sequence region is searched for the sequence motif AA (N19), where N designates a nucleotide. The resulting siRNA sequence has 19 nucleotides following the motif AA and preferably additionally an additional uridine or thymidine residue. In the case of synthetic siRNA oligonucleotides, the uridine residue may preferably be replaced by thymidine.

  In other approaches, guidelines by Reynolds et al. May be used.

Reynold et al. Have proposed the following criteria for selecting potential shRNA or siRNA target sequences:
1 30-50% guanine-cytosine content 2 at least 3 adenine or uracil 3 at positions 15-19 of the sense strand 3 lack of hairpin structure between molecules 4 adenine 5 at sense strand position 19 sense strand position Adenine at 3 6 Uracil at position 10 of the sense strand 7 No guanine or cytosine at position 19 of the sense strand 8 No guanine at position 13 of the sense strand

These eight criteria are weighted by the following scheme:
(I) 1 point for reference 1, 3, 4, 5 and 6 (ii) 1 point for each adenine or uridine at position 3 to 19 corresponding bases (position 2)
(Iii) 1 point each when the standard 7-8 is not achieved.

  According to Reynolds, only siRNA or shRNA sequences should be considered to have a point value of at least 6 according to this scheme. Such siRNA sequences are used for correspondence searches using the BLAST program. Following this approach, the siRNA sequence leads to a target structure with no special repression.

  If siRNA or shRNA sequences are thus identified, they may be cloned into an expression plasmid. Thus, the RNA sequence is cloned into plasmid p suppression (pSHH, Imgenex, San Diego, Calif.). In order to clone the siRNA sequence into the pSHH construct, hybridized DNA oligonucleotides with siRNA sense sequences, spacers, corresponding antisense sequences and stop sequences can be used.

In a preferred embodiment, the present invention is a non-invasive method for monitoring in vivo expression of a gene of interest in a cell, comprising:
a)
aa) a promoter sequence;
bb) a sequence encoding the gene of the polypeptide of interest operably linked to the promoter sequence;
cc) inducing into the cell a nucleic acid molecule having a sequence encoding a fluorescent reporter polypeptide operatively fused to the sequence of bb);
b) detecting the fluorescent reporter polypeptide in the cell by a non-invasive optical imaging method;
c) contacting the cell with RNAi adjusted for expression of the gene of interest;
d) detecting the fluorescent reporter polypeptide in the cell by a non-invasive optical imaging method during the step c);
e) comparing the level of the fluorescent reporter polypeptide detected in step b) with the level of the fluorescent reporter polypeptide detected in step d);
and f) assigning a decrease in the expression of the gene of interest in the subject induced by RNAi based on the comparison in step e).

In another preferred embodiment, the present invention provides:
a)
aa) a promoter sequence;
bb) a sequence encoding the gene of the polypeptide of interest operably linked to the promoter sequence;
cc) directing a nucleic acid molecule having a sequence encoding a fluorescent reporter polypeptide operatively fused to the sequence of bb) into a cell outside the human body or animal body;
b) detecting the fluorescent reporter polypeptide in the cell by a non-invasive optical imaging method;
c) contacting the cell with RNAi tuned for the gene of interest;
d) detecting the fluorescent reporter polypeptide in the cell by a non-invasive optical imaging method during the step c);
e) comparing the level of the fluorescent reporter polypeptide detected in step b) with the level of the fluorescent reporter polypeptide detected in step d);
and f) assigning a decrease in the expression of the gene of interest in the subject induced by RNAi based on the comparison in step e).

  In the context of monitoring the effect of RNAi in cells outside the human or animal body, one embodiment of the present invention relates to a method of targeting RNAi in step c) to a cell that has already expressed a reporter polypeptide, and thus a nucleic acid. The molecule has already been introduced into the cell. The method wherein the second coding sequence is operably linked to a strong constitutive promoter includes the second nucleic acid molecule having the second coding sequence or the second on the first nucleic acid molecule. Have the coding sequence.

  In any method according to the invention that refers to RNAi, the gene of interest sequence of the nucleic acid molecule used in the method comprises at least a region of the gene of interest to which RNAi is regulated.

  In both in vivo and in vitro situations, the present invention describes in a preferred embodiment a method in which the amount of RNAi is increased or decreased by allocation in step f) as listed above. Furthermore, the present invention provides that, in a preferred embodiment, the different RNAi adjusted for one gene of interest is the most of the expression of said gene of interest by assignment in step f) as listed above. Describes methods that are tested separately or in combination to obtain a powerful increase.

  In other embodiments, the present invention relates to methods and / or use of nucleic acids as described above.

  In one embodiment relating to an in vivo setup, the present invention is for monitoring, for example, an increase in expression of a gene of a subject induced by a demethylating drug over time in vivo, for example when treating a disease with a demethylating drug. Regarding the use of the method. Thus, a method for monitoring the therapeutic effect of demethylating drugs is used. Similarly, the present invention, in another embodiment, relates to the use of a method for monitoring a decrease in gene expression of a subject induced by RNAi over time in vivo when treating a disease with RNAi. Here, a method for monitoring the therapeutic effect of RNAi is used. Obviously, in the case of RNAi, the first detection step produces a signal with the optical imaging method, otherwise no decrease can be detected. Thus, if there is no reporter polypeptide detected in the first detection step, the situation of the preceding step is optimized to find the optimal situation where detection is possible. Only then is the therapeutic compound administered.

  When a functional gene of a subject polypeptide is expressed by a nucleic acid molecule of the invention, the functional gene of the subject polypeptide is used as a therapy. As outlined above, for example, in the case of gene silencing in cancer, it is often found that tumor suppressor genes should be silenced. This is treated with a demethylating agent or an HDAC response inhibitor and monitored by the method of the present invention. When using the method, a functional tumor suppressor polypeptide is introduced encoded by a gene of interest sequence to exert an additional positive therapeutic effect.

  Also, in one embodiment, the method according to the invention is used to adjust the amount of demethylating drug or RNAi in order to obtain an increase or decrease in the expression of the gene of interest. Furthermore, methods of testing different methylating agents or RNAi are used to identify the most potent demethylating agent / RNAi, either separately or in combination with other demethylating agents and RNAi, respectively. This applies to both in vivo and in vitro situations. In an in vitro setup such as a cell culture system as described above, the method of the invention is preferably used to screen for compounds that have an effect on altered expression of a symmetric gene. Thus, the method is used as a fast screening system for testing drugs that can affect gene expression.

  The nucleic acid molecule or method according to the present invention, in one embodiment, not only monitors the delivery of a drug and simultaneously monitors the increase in expression of a gene of interest induced by said drug, but also for the delivery of a demethylated drug. Used for. Similarly, a nucleic acid molecule or method according to the present invention, in another embodiment, delivers RNAi and at the same time not only monitors RNAi-induced decrease in the expression of the gene of interest, but also monitors the delivery of said RNAi. Used to do. This also applies in both in vivo and in vitro situations.

Other preferred embodiments of the invention relate to the following.
1. A non-invasive method for monitoring the expression of a gene of interest in a cell,
a)
aa) a promoter sequence;
bb) a sequence encoding the gene of the polypeptide of interest operably linked to the promoter sequence;
cc) inducing into the cell a nucleic acid molecule having a sequence encoding a fluorescent reporter polypeptide operatively fused to the sequence of bb);
b) detecting the fluorescent reporter polypeptide in the cell by a non-invasive optical imaging method;
c) contacting the cell with a compound that affects the expression of the gene of interest;
d) detecting the fluorescent reporter polypeptide in the cell by a non-invasive optical imaging method during the step c);
e) comparing the level of the fluorescent reporter polypeptide detected in step b) with the level of the fluorescent reporter polypeptide detected in step d);
f) assigning at least a change in the expression of the gene in the subject induced by the compound based on the comparison in step e).

2. The method according to 1 above, wherein the expression of a target gene in a cell is monitored non-invasively in the body,
a)
aa) a methylated promoter sequence;
bb) a sequence encoding the gene of the polypeptide of interest operably linked to the promoter sequence;
cc) inducing into the cell a nucleic acid molecule having a sequence encoding a fluorescent reporter polypeptide operatively fused to the sequence of bb);
b) detecting the fluorescent reporter polypeptide in the cell by a non-invasive optical imaging method;
c) contacting the cell with a demethylating agent;
d) detecting the fluorescent reporter polypeptide in the cell by a non-invasive optical imaging method during the step c);
e) comparing the level of the fluorescent reporter polypeptide detected in step b) with the level of the fluorescent reporter polypeptide detected in step d);
and f) assigning at least an increase in the expression of the gene in the subject induced by the demethylating agent based on the comparison in step e).

3. a)
aa) a methylated promoter sequence;
bb) a sequence encoding the gene of the polypeptide of interest operably linked to the promoter sequence;
cc) directing a nucleic acid molecule having a sequence encoding a fluorescent reporter polypeptide operatively fused to the sequence of bb) into a cell outside the human body or animal body;
b) detecting the fluorescent reporter polypeptide in the cell by a non-invasive optical imaging method;
c) contacting the cell with a demethylating agent;
d) detecting the fluorescent reporter polypeptide in the cell by a non-invasive optical imaging method during the step c);
e) comparing the level of the fluorescent reporter polypeptide detected in step b) with the level of the fluorescent reporter polypeptide detected in step d);
f) assigning an increase in the expression of the gene of interest in the subject induced by the demethylating agent based on the comparison in step e) at least.

4). The method according to 1 above, wherein the expression of a target gene in a cell is monitored non-invasively in the body,
a)
aa) a promoter sequence;
bb) a sequence encoding a non-functional polypeptide of the gene of interest operably linked to the promoter sequence;
cc) inducing into the cell a nucleic acid molecule having a sequence encoding a fluorescent reporter polypeptide operatively fused to the sequence of bb);
b) detecting the fluorescent reporter polypeptide in the cell by a non-invasive optical imaging method;
c) contacting the cell with RNAi tuned for the gene of interest;
d) detecting the fluorescent reporter polypeptide in the cell by a non-invasive optical imaging method during the step c);
e) comparing the level of the fluorescent reporter polypeptide detected in step b) with the level of the fluorescent reporter polypeptide detected in step d);
f) assigning at least an increase in the expression of the gene of interest in the subject induced by the RNAi based on the comparison in step e).

5. a)
aa) a promoter sequence;
bb) a sequence encoding the gene of the polypeptide of interest operably linked to the promoter sequence;
cc) directing a nucleic acid molecule having a sequence encoding a fluorescent reporter polypeptide operatively fused to the sequence of bb) into a cell outside a human or animal body;
b) detecting the fluorescent reporter polypeptide in the cell by a non-invasive optical imaging method;
c) contacting the cell with RNAi tuned for the gene of interest;
d) detecting the fluorescent reporter polypeptide in the cell by a non-invasive optical imaging method during the step c);
e) comparing the level of the fluorescent reporter polypeptide detected in step b) with the level of the fluorescent reporter polypeptide detected in step d);
f) assigning an increase in the expression of the gene in the subject induced by the RNAi based on the comparison in step e) at least.

  6). The nucleic acid molecule and / or the RNAi are introgressed with the help of sonoporation, localized injection, or liposome bags that carry antibodies to host cell antigens for correct targeting, identification and localization or a mixture thereof 6. The method according to any one of 1 to 5 above, wherein the method is introduced into the cell by:

  7). a) a methylated promoter sequence; b) an operably linked sequence encoding a gene of interest polypeptide; and an operably fused sequence encoding a fluorescent reporter polypeptide, An isolated nucleic acid molecule wherein the coding sequence consisting of b) and c) above has a single start codon at the 5 'end and a single stop codon at the 3' end.

  8). 8. The nucleic acid gene according to 7, wherein the gene of interest is a tumor suppressor gene or a gene suspected of causing a disease due to silencing.

  9. a) a promoter sequence, b) an operably linked sequence encoding a non-functional polypeptide of the gene of interest, and an operably fused sequence encoding a fluorescent reporter polypeptide; An isolated nucleic acid molecule, wherein the coding sequence consisting of b) and c) has a single start codon at the 5 'end and a single stop codon at the 3' end.

  10. The gene of the sequence of interest encoding a non-functional polypeptide is part of the entire gene of the sequence of interest or at least one insertion, at least one deletion to obtain a non-functional gene of the polypeptide of interest 10. The nucleic acid molecule according to 9, wherein the nucleic acid molecule has any one of all genes of the sequence of interest including at least one nucleotide exchange, or a combination thereof, and the gene of the sequence of interest does not have a stop codon.

  11. The nucleic acid molecule according to 9 and 10, wherein the gene of interest is an oncogene, proto-oncogene, or a gene suspected of causing a disease due to overexpression.

  12 Any one of 1 to 6 for monitoring the expression of the gene of interest in the cell over time when contacting the cell with a compound that affects the expression of the gene of interest, and thus monitoring the effect of the compound. Use of the method according to paragraph.

  13. Sending a compound that affects the expression of a gene of interest in a cell and not only monitoring the influence of the expression of the gene of interest in the subject induced by the compound, but simultaneously monitoring the delivery of the compound Use of the method according to any one of 6 above.

  14 Use of the method according to any one of 1 to 6 above to adjust the amount of a compound that affects the expression of the gene of interest in order to obtain other changes in the expression of the gene of interest.

  15. Use of the method according to 2 and 3 above to test different demethylating agents separately or in combination to identify the most potent demethylating agents.

  16. 6. Use of the method according to 4 and 5 above to test different RNAi that are regulated against one gene of interest in order to identify the most potent RNAi separately or in combination.

  17. The nucleic acid molecule according to any of 7 to 11 for monitoring the expression of a gene of interest in a cell over time when contacting the cell with a compound that affects the expression of the gene of interest, and thus monitoring the effect of the compound Use of.

  13. 7 for delivering a compound that affects the expression of a gene of interest in a cell and simultaneously monitoring the delivery of the compound as well as monitoring the effect of the expression of the gene on the subject induced by the compound Use of the nucleic acid molecule according to any one of 1 to 11.

  14 Use of the nucleic acid molecule according to any of 7 to 11 above to adjust the amount of a compound that affects the expression of the gene of interest in order to obtain other changes in the expression of the gene of interest.

  15. Use of a nucleic acid molecule according to any of the above 7 to 11 to test different demethylating agents in order to identify the most potent demethylating agents separately or in combination.

  16. Use of a nucleic acid molecule according to any of 7 to 11 above to test different RNAi that are adjusted for one gene of interest in order to identify the most potent RNAi separately or in combination.

  It will be understood that the examples and drawings are not to be construed as limiting. Those skilled in the art can clearly grasp further variations of the principles present in the present application.

  When RNAi adjusted for Her-2neu is transferred into breast cancer tissue, expression of Her-2neu in the breast cancer tissue (a gene known to be overexpressed in breast cancer) is non-invasive. Protocol for how to monitor with

  A schematic diagram of the steps is shown in FIG.

Step 1:
A DNA construct is prepared. The construct has a human promoter known to be active in breast cancer tissue. The operably linked promoter is a coding region. This coding region consists of the Her-2neu gene fused to the gene for a fluorescent protein (eg GFP). The Her-2neu sequence is modified such that a non-functional polypeptide of Her-2neu is expressed, for example, by a frameshift mutation. At the 3 'end, the sequence of the fluorescent protein gene is fused to the Her-2neu sequence so that the resulting protein is transcribed as a single fusion protein and the resulting fluorescent polypeptide is functional. This is because in the current example, the frameshift mutation induced in the Her-2neu sequence is induced at the beginning of sequence coding for the fluorescent protein in order to encode a functional fluorescent protein such as GFP. Means compensated by shift mutation.

Step 2:
The composition as described above can be introduced into a breast cancer tissue, for example, by injecting a liposome bag carrying the composition, preferably an antibody directed against an antigen of breast cancer cells for the purpose of targeting. Is done. It should be noted that this targeting step is beneficial for the next visualization step, as only cancer-affected tissue is detected. However, this is also achieved when the above-described promoter is functional only in breast cancer cells and thus the coding region (and thus the fluorescent protein) is expressed only in these cells. In either case, the fusion gene is expressed in cancerous cells that have a functional fluorescent protein domain, eg, GFP as well as a non-functional domain of Her-2neu.

Step 3:
The fluorescent protein domain is visualized and thus the breast cancer tissue glows. This is done by using an optical imaging device that excites the fluorescent protein at a specific wavelength followed by detection of light emitted at the corresponding wavelength. Ultraviolet light is used to excite GFP followed by detection of light emitted as green. Preferably, compounds that are excited by infrared and / or compounds such as near-infrared fluorescent proteins may be used (see, for example, “Nature Methods 4, 741-746 (2007)” by Shellabo Day et al. ing).

Step 4:
Double-stranded RNAi tailored to Her-2neu is introgressed into breast cancer tissue. This is again done by injecting a liposome bag carrying the RNAi construct, preferably an antibody directed against the breast cancer cell antigen for targeting purposes.

Step 5:
Similar to the expression of the endogenous Her-2neu gene, the expression of the fusion gene introduced in step 2 is blocked. This is achieved by RNAi that selectively blocks translation from the corresponding mRNA. In order to fuse the Her-2neu gene to the sequence coding for the fluorescent protein, the expression of the fluorescent protein is also blocked. Thus, there is no or only a small amount of fluorescent protein in breast cancer tissue upon RNAi gene transfer.

Step 6:
Again, the fluorescent protein domain is visualized in the corresponding tissue. As mentioned above, ultraviolet light is used to excite GFP followed by detection of light emitted as green. Preferably, a compound excited by infrared rays may be used. In the case of the entire translation block of mRNA transcribed from the fusion gene by the introduced RNAi, the fluorescent protein cannot be detected. Thus, breast cancer tissue can no longer be visualized. In the case of disease, the signal is weak compared to the light detected in step 3. Overall, this strongly suggests that endogenous Her-2neu was also blocked, thus achieving tumor regression.

  In summary, the therapeutic effect of RNAi is monitored in vivo in breast cancer tissue in a non-invasive manner.

Claims (15)

A non-invasive method for monitoring the expression of a gene of interest in a cell,
a)
aa) a promoter sequence;
bb) a sequence encoding the gene of the polypeptide of interest operably linked to the promoter sequence;
cc) inducing into the cell a nucleic acid molecule having a sequence encoding a fluorescent reporter polypeptide operatively fused to the sequence of bb);
b) detecting the fluorescent reporter polypeptide in the cell by a non-invasive optical imaging method;
c) contacting the cell with a compound that affects the expression of the gene of interest;
d) detecting the fluorescent reporter polypeptide in the cell by a non-invasive optical imaging method during the step c);
e) comparing the level of the fluorescent reporter polypeptide detected in step b) with the level of the fluorescent reporter polypeptide detected in step d);
f) assigning at least a change in the expression of the gene in the subject induced by the compound based on the comparison in step e). The method according to claim 1, wherein the expression of the gene of interest in the cell is monitored non-invasively in the body,
a)
aa) a methylated promoter sequence;
bb) a sequence encoding the gene of the polypeptide of interest operably linked to the promoter sequence;
cc) inducing into the cell a nucleic acid molecule having a sequence encoding a fluorescent reporter polypeptide operatively fused to the sequence of bb);
b) detecting the fluorescent reporter polypeptide in the cell by a non-invasive optical imaging method;
c) contacting the cell with a demethylating agent;
d) detecting the fluorescent reporter polypeptide in the cell by a non-invasive optical imaging method during the step c);
e) comparing the level of the fluorescent reporter polypeptide detected in step b) with the level of the fluorescent reporter polypeptide detected in step d);
and f) assigning at least an increase in the expression of the gene in the subject induced by the demethylating agent based on the comparison in step e). a)
aa) a methylated promoter sequence;
bb) a sequence encoding the gene of the polypeptide of interest operably linked to the promoter sequence;
cc) directing a nucleic acid molecule having a sequence encoding a fluorescent reporter polypeptide operatively fused to the sequence of bb) into a cell outside the human body or animal body;
b) detecting the fluorescent reporter polypeptide in the cell by a non-invasive optical imaging method;
c) contacting the cell with a demethylating agent;
d) detecting the fluorescent reporter polypeptide in the cell by a non-invasive optical imaging method during the step c);
e) comparing the level of the fluorescent reporter polypeptide detected in step b) with the level of the fluorescent reporter polypeptide detected in step d);
f) assigning an increase in the expression of the gene of the subject induced by the demethylating agent based on the comparison in step e). The method according to claim 1, wherein non-invasive monitoring of in vivo expression of a gene of interest in a cell is provided,
a)
aa) a promoter sequence;
bb) a sequence encoding a non-functional polypeptide of the gene of interest operably linked to the promoter sequence;
cc) inducing into the cell a nucleic acid molecule having a sequence encoding a fluorescent reporter polypeptide operatively fused to the sequence of bb);
b) detecting the fluorescent reporter polypeptide in the cell by a non-invasive optical imaging method;
c) contacting the cell with RNAi tuned for the gene of interest;
d) detecting the fluorescent reporter polypeptide in the cell by a non-invasive optical imaging method during the step c);
e) comparing the level of the fluorescent reporter polypeptide detected in step b) with the level of the fluorescent reporter polypeptide detected in step d);
f) assigning at least a decrease in the expression of the gene of interest in the subject induced by the RNAi based on the comparison in step e). a)
aa) a promoter sequence;
bb) a sequence encoding the gene of the polypeptide of interest operably linked to the promoter sequence;
cc) directing a nucleic acid molecule having a sequence encoding a fluorescent reporter polypeptide operatively fused to the sequence of bb) into a cell outside a human or animal body;
b) detecting the fluorescent reporter polypeptide in the cell by a non-invasive optical imaging method;
c) contacting the cell with RNAi tuned for the gene of interest;
d) detecting the fluorescent reporter polypeptide in the cell by a non-invasive optical imaging method during the step c);
e) comparing the level of the fluorescent reporter polypeptide detected in step b) with the level of the fluorescent reporter polypeptide detected in step d);
f) assigning a decrease in the expression of the gene of interest in the subject induced by the RNAi based at least on the comparison in step e).   The nucleic acid molecule and / or the RNAi are introgressed with the help of sonoporation, localized injection, or liposome bags that carry antibodies to host cell antigens for correct targeting, identification and localization or a mixture thereof The method according to any one of claims 1 to 5, wherein the method is introduced into the cell by the method. a) a methylated promoter sequence;
b) CADM1 (SEQ ID No. 2), Rb (SEQ ID No. 1), ZMYND10 (SEQ ID No. 3), RASSF5 (SEQ ID No. 4), PTEN operatively linked to the promoter sequence Encodes a polypeptide selected from the group of polypeptides having (SEQ ID No. 5), SERPINB5 (SEQ ID No. 6), EPB41L3 (SEQ ID No. 7), and DAPK1 (SEQ ID No. 8) An array to
c) an isolated nucleic acid molecule having a sequence encoding a fluorescent reporter polypeptide operably fused to the sequence of b), wherein the coding sequence consisting of b) and c) is 5 A nucleic acid molecule having a single start codon at the 'end and a single stop codon at the 3' end. a) a promoter sequence;
b) VEGF (SEQ ID No. 10), RAS (SEQ ID No. 11), Wnt (SEQ ID No. 12), MYC (SEQ ID No. 13), ERK operably linked to the promoter sequence. A sequence encoding a non-functional polypeptide selected from the group of polypeptides having (SEQ ID No. 14) and TRK (SEQ ID No. 15);
c) an isolated nucleic acid molecule having a sequence encoding a fluorescent reporter polypeptide operatively fused to the sequence of b), wherein the coding sequence consisting of b) and c) is 5 ′ A nucleic acid molecule having a single start codon at the end and a single stop codon at the 3 'end.   VEGF (SEQ ID No. 10), RAS (SEQ ID No. 11), Wnt (SEQ ID No. 12), MYC (SEQ ID No. 13), ERK (SEQ ID No. 14) and TRK (SEQ ID No. 14) .15) a sequence encoding a non-functional polypeptide selected from the group of polypeptides having a portion of the entire sequence, or at least one insertion, at least one to obtain a non-functional polypeptide 9. The nucleic acid molecule of claim 8, having any of a deletion, at least one nucleotide exchange, or an entire sequence including a combination thereof, wherein the sequence has no stop codon. a) a promoter sequence;
b) Her-2neu (SEQ ID No. 9) comprising at least one insertion, at least one nucleic acid exchange, or a combination thereof to obtain a non-functional polypeptide operatively linked to the promoter sequence A sequence encoding a non-functional polypeptide of the sequence without a stop codon;
c) an isolated nucleic acid molecule having a sequence encoding a fluorescent reporter polypeptide operably fused to the sequence of b), wherein the coding sequence consisting of b) and c) is 5 A nucleic acid molecule having a single start codon at the 'end and a single stop codon at the 3' end.   11. A method as claimed in any one of claims 7 to 10 for monitoring the expression of the gene of interest within the cell over a period of time when the cell is contacted with a compound that affects expression of the gene of interest, and thus monitoring the effect of the compound. Use of the nucleic acid molecule according to claim 1 or the method according to any one of claims 1 to 6.   Claims for delivering a compound that affects the expression of a gene of interest in a cell and simultaneously monitoring the delivery of said compound as well as monitoring the effect on expression of said gene of interest in said subject by said compound Use of the nucleic acid molecule according to any one of claims 7 to 10 or the method according to any one of claims 1 to 6.   11. The nucleic acid molecule or claim 1 for adjusting the amount of a compound that affects the expression of said gene of interest in order to obtain other changes in the expression of the gene of interest. Use of the method according to any one of 1 to 6.   Use of the nucleic acid molecule according to claim 7 or the method according to claims 2 and 3 for testing different demethylating drugs, separately or in combination, in order to identify the most potent demethylating drugs.   11. A nucleic acid molecule according to any one of claims 8 to 10 for testing different RNAi that are adjusted for one gene of interest in order to identify the most potent RNAi separately or in combination. Or use of the method according to claims 4 and 5.

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